Synaptogenesis in the developing rat central nervous system occurs primarily during the first three weeks of postnatal life. At present, we only have a rudimentary understanding of (i) the molecular constituents of synaptic junctions, (ii) in what form synaptic proteins are correctly sorted and trafficked to newly forming synapses, and (iii) how junctional proteins are able to define the various pre and postsynaptic subdomains. For example, the presynaptic terminal is a highly specialized cellular compartment designed to rapidly and efficiently release neurotransmitter from synaptic vesicles. Much progress has been made in understanding the molecular machinery involved in the Ca+2 regulated exocytosis of SVs at the presynaptic plasma membrane, as well as the endocytotic events that lead to the recycling of SV membranes. Knowledge is limited, however, concerning how the docking, fusion and recycling of SVs is restricted to the active zone. EM studies of nerve terminals have shown that the cytoplasm surrounding the readily releasable pool of SVs is composed of an electron dense network of fine filaments referred to as the junctional cytoskeletal matrix. In many respects, this presynaptic cytoskeletal matrix is the structural equivalent to that found at the PSD and is thought to hold the PSD and active zone in register. It may also help define the active zone by clustering components of the exo and endocytotic machinery. At present, little is known about the exact nature of the proteins that define this region, named the presynsptic junction (PSJ). Preliminary analysis of structural components of synaptic junctions has led to the identification of two novel structurally related presynaptic cytoskeletal matrix proteins of the PSJ, Piccolo and Bassoon. Biochemical, cellular and functional studies strongly indicate that they are structural elements of the PSJ and have led to the hypotheses that they play a fundamental role in defining the active zone. Experiments outlined in this application will test this hypothesis for Piccolo. Moreover, the investigator will test the hypotheses that the central region of Piccolo is involved in anchoring it in the PSJ and that components of the PSJ are sorted and trafficked to nerve terminals as a pre-PSJ particle. These studies will provide an entry point to understanding the molecules and mechanisms underlying PSJ assembly.